Outline

Detection of disseminated tumour cells (DTC) in histopathologically inconspicuous tissues is presumably of high importance but often flawed with the difficulty to discover one mutated cell in a surplus of wild-type cells. The aim of this study was to develop a rapid and sensitive assay for detecting K-ras codon 12 mutations indicating DTC in tissue samples from colorectal carcinoma patients.

Methods: The double enriched nested (DEN-) PCR makes use of the thermostable restriction enzyme BstNI during the first PCR and locked nucleic acid (LNA) clamping oligonucleotides during the second PCR. Both PCR steps provide for suppression of the K-ras wild-type allele thus allowing a highly selective amplification of a K-ras codon 12 mutated sequence. All samples underwent cycle sequencing to assess the genomic sequence. Tissue samples were obtained from 49 colorectal carcinoma patients.

Results: First, standard cell line mixtures (SW620 and SKW3) were used to identify the detection limit of the novel DEN-PCR. The dilutions ranged from 1 mutant in 10 to 106 wild-type cells. Current PCR assays are either less sensitive (Restriction Enzyme Mediated Selective [REMS-] PCR: 1 mutant in 102 wild type cells) or time consuming (Restriction Fragment Length Polymorphism [RFLP-] PCR: 36 hours). DEN-PCR showed a sensitivity of 1 mutated cell in 106 – 107 wild type cells, its performance took around 8 hours, thus ranking within the most rapid assays.

Then, tissue samples from colon carcinoma patients were tested for assessing the clinical applicability of the assay. Out of 49 colorectal carcinoma patients, 25 (51%) tumour samples were found to be mutated in K-ras codon 12. All mutations could be confirmed by cycle sequencing. Liver tissue harboured K-ras codon 12 mutations in 8 of 25 (32%) samples, lymph node tissue in 8 of 25 (32%) samples and bone marrow in 4 of 25 (16%) samples. The most frequent mutation in liver tissue was G12N (5 of 8), those in lymph node tissue were G12N (3 of 8) and G12S (3 of 8). Overall, more DTC were found with DEN-PCR than with standard methods (RFLP-, REMS-, Real Time PCR with PNA).

Conclusion: The DEN-PCR assay is more rapid and sensitive than others and shows high reliability as shown by sequencing. Therefore, this assay has clear potential for detecting DTC.